Many bacterial pathogens utilize toxins to cause serious disease. For instance, anthrax lethal toxin (LeTx) is one of the primary virulence factors that Bacillus anthracis utilizes to subvert the host immune system and induce cell death, resulting in a rapid onset of death that is often untreatable with modern medicine, as demonstrated by the 45 percent case-fatality rate among patients with inhalation anthrax during the 2001 attacks. There is an urgent need for a better understanding of the mechanism of anthrax LeTx and corresponding drugs to inhibit these processes. I propose to identify host cellular factors required for toxin entry and toxin-induced cell death using a LeTx macrophage viability model and the complementary approaches of chemical genetics and RNAi. I will conduct a chemical high-throughput screen in macrophages to identify small molecule inhibitors of LeTx-induced cell death. I will prioritize inhibitors according to their potency and activities in secondary assays designed to determine what host pathways or events are inhibited, such as those involved in toxin cell entry or toxicity once the toxin enters the cell. Small molecules with known host targets will be further evaluated using RNAi and structurally different compounds that target the same protein. Alternatively, the best compounds with no known target will be applied to affinity chromatography to identify their host targets, which will help determine their mechanisms of action. We have successfully conducted pilot chemical and RNAi HTS assays that have identified promising leads, indicating that larger screens will likely succeed. The long-term goal of this application is to gain insight into the general biology of toxin translocation and toxicity mechanisms, and the specific biology of anthrax toxin. Accomplishing the proposed aims will illuminate the molecular basis of LeTx entry and toxicity in macrophages and yield small molecules that will serve both as tools to probe LeTx biology as well as proof-of-concept leads for the development of novel therapeutics. This research may lead to new paradigms in host-targeted therapeutics that circumvent the growing problem of antibiotic resistance of important infectious diseases. PUBLIC HEALTH RELEVANCE: Many bacterial pathogens produce toxins that cause serious disease including cholera, tetanus, botulism, diphtheria, and anthrax and often share key steps in cell entry and cell death processes that rely on host proteins. I propose to use an anthrax lethal toxin-induced cell death model to identify host proteins and pathways co-opted by toxins. This work will illuminate the molecular mechanisms underlying resultant pathology and may provide clues for how to combat toxin-related diseases.